TAD boundary deletion causes PITX2-related cardiac electrical and structural defects

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.


Recruitment
Inclusion criteria were families presenting forms of sinus dysfunction defined as the presence of sinus bradycardia (daytime heart rate <50 BPM), diurnal sinus pause >3 seconds, or chronotropic incompetence objectified during an exercise test; cardiac morphological abnormalities such as atrial septal defect, left ventricular non-compaction, mitral valve prolapse or billowing, or an abnormal systemic venous return and other cardiac rhythm disorders and in particular atrial fibrillation or ECG abnormalities

Ethics oversight
The study was conducted according to international recommendations.Written consent was systematically obtained before clinical and genetic analysis was performed in these patients.This research project has been approved by the French Minister of education and research as well as by the personal protection committee (DC-2011(DC- -1399)).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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We looked for a maximum of independant families presenting a similar phenotype.All families member of each families are recruted.Data exclusions Individual refusing the consent have been excluded from the study Replication 7 independant families have been recruited and analysed separately.
Mouse model : all attempts at replication were successful Randomization We included all family members belonging to families presenting a similar phenotype.
Mouse model : Mice were allocated into experimental groups based on their genotype.Both male and female mice were used.

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histone H3K4 (H3K4me3, 1:50, CST#9751T) and the negative control antibody mAb IgG isotype (1:50, CST#66362).Antibodies mAb against acetyl H3K27 (H3K27ac, 1:100, CST#39133), Monomethylated H3K4 (H3K4me1, 1:50, CST#39297) and CTCF (1:50, CST#61311) Validation CST antibodies used adhere to the Hallmarks of Antibody Validation (https://www.cellsignal.com/about-us/cst-antibody-validationprinciples)Eukaryotic cell lines Policy information about cell lines and Sex and Gender in Research Cell line source(s) Provide detail on software version and revision number and on specific parameters(model/functions, brain extraction,  segmentation, smoothing kernel size, etc.).If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g.original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.
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